Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.     

Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.

Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.     

Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.     

The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.     

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.     

Neuronal NOS-mediated nitration and inactivation of manganese superoxide dismutase in brain after experimental and human brain injury

Manganese superoxide dismutase (MnSOD) provides the first line of defense against superoxide generated in mitochondria. SOD competes with nitric oxide for reaction with superoxide and prevents generation of peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. Thus, sufficient amounts of catalytically competent MnSOD are required to prevent mitochondrial damage. Increased nitrotyrosine immunoreactivity has been reported after traumatic brain injury (TBI); however, the specific protein targets containing modified tyrosine residues and functional consequence of this modification have not been identified. In this study, we show that MnSOD is a target of tyrosine nitration that is associated with a decrease in its enzymatic activity after TBI in mice. Similar findings were obtained in temporal lobe cortical samples obtained from TBI cases versus control patients who died of causes not related to CNS trauma. Increased nitrotyrosine immunoreactivity was detected at 2 h and 24 h versus 72 h after experimental TBI and co-localized with the neuronal marker NeuN. Inhibition and/or genetic deficiency of neuronal nitric oxide synthase (nNOS) but not endothelial nitric oxide synthase (eNOS) attenuated MnSOD nitration after TBI. At 24 h after TBI, there was predominantly polymorphonuclear leukocytes accumulation in mouse brain whereas macrophages were the predominant inflammatory cell type at 72 h after injury. However, a selective inhibitor or genetic deficiency of inducible nitric oxide synthase (iNOS) failed to affect MnSOD nitration. Nitration of MnSOD is a likely consequence of peroxynitrite within the intracellular milieu of neurons after TBI. Nitration and inactivation of MnSOD could lead to self-amplification of oxidative stress in the brain progressively enhancing peroxynitrite production and secondary damage.     

Estradiol attenuates high glucose-induced endothelial nitrotyrosine: role for neuronal nitric oxide synthase

Hyperglycemia in diabetes causes increased oxidative stress in the vascular endothelium with generation of free radicals such as superoxide. Peroxynitrite, a highly reactive species generated from superoxide and nitric oxide (NO), induces proinflammatory tyrosine nitration of intracellular proteins under such conditions. The female sex hormone estrogen appears to exert protective effects on the nondiabetic endothelium. However, several studies show reduced vascular protection in women with diabetes, suggesting alterations in estrogen signaling under high glucose. In this study, we examined the endothelial effects of estrogen under increasing glucose levels, focusing on nitrotyrosine and peroxynitrite. Human umbilical vein endothelial cells were incubated with normal (5.5 mM) or high (15.5 or 30.5 mM) glucose before addition of estradiol (E2, 1 or 10 nM). Selective NO synthase (NOS) inhibitors were used to determine the role of specific NOS isoforms. Addition of E2 significantly reduced high glucose-induced increase in peroxynitrite and consequently, nitrotyrosine. The superoxide levels were unchanged, suggesting effects on NO generation. Inhibition of neuronal NOS (nNOS) reduced high glucose-induced nitrotyrosine, demonstrating a critical role for this enzyme. E2 increased nNOS activity under normal glucose while decreasing it under high glucose as determined by its phosphorylation status. These data show that nNOS contributes to endothelial peroxynitrite and subsequent nitrotyrosine generation under high glucose, which can be attenuated by E2 through nNOS inhibition. The altered regulation of nNOS by E2 under high glucose is a potential therapeutic target in women with diabetes.     

The impact of metal catalysis on protein tyrosine nitration by peroxynitrite

In a series of heme and non-heme proteins the nitration of tyrosine residues was assessed by complete pronase digestion and subsequent HPLC-based separation of 3-nitrotyrosine. Bolus addition of peroxynitrite caused comparable nitration levels in all tested proteins. Nitration mainly depended on the total amount of tyrosine residues as well as on surface exposition. In contrast, when superoxide and nitrogen monoxide (NO) were generated at equal rates to yield low steady-state concentrations of peroxynitrite, metal catalysis seemed to play a dominant role in determining the sensitivity and selectivity of peroxynitrite-mediated tyrosine nitration in proteins. Especially, the heme-thiolate containing proteins cytochrome P450(BM-3) (wild type and F87Y variant) and prostacyclin synthase were nitrated with high efficacy. Nitration by co-generated NO/O(2)(-) was inhibited in the presence of superoxide dismutase. The NO source alone only yielded background nitration levels. Upon changing the NO/O(2)(-) ratio to an excess of NO, a decrease in nitration in agreement with trapping of peroxynitrite and derived radicals by NO was observed. These results clearly identify peroxynitrite as the nitrating species even at low steady-state concentrations and demonstrate that metal catalysis plays an important role in nitration of protein-bound tyrosine.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.     

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: modeling the impact of superoxide dismutase and nitric oxide diffusion

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO(-)) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O(2)*(-)) and nitric oxide ((*)NO), are formed at ratios (O(2)*(-)/(*)NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of (*)NO preclude accumulation of O(2)*(-) and (*)NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation (J(NO(2(-))Y)) during exposure to simultaneous fluxes of superoxide (J(O(2)*(-))) and nitric oxide (J((*)NO)), varying the radical flux ratios (J(O(2)*(-))/ J((*)NO)), in the presence of carbon dioxide. With a basic set of reactions, J(NO(2(-))Y) as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and (*)NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J(NO(2(-))Y) as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O(2)*(-) or (*)NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.     

Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins

Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Role of superoxide-nitric oxide interactions in the accelerated age-related loss of muscle mass in mice lacking Cu,Zn superoxide dismutase

Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age-related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1(-/-) mice were loaded with NO-sensitive (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3-nitrotyrosine (3-NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1(-/-) mice compared with those from WT mice. Fibers from Sod1(-/-) mice had decreased NO availability (decreased DAF-FM fluorescence), increased 3-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1(-/-) mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1(-/-) mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1(-/-) mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.     

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.     

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.